Abstract Despite the importance of extracellular gradients in the formation and maintenance of tissue formation, there are few analytical tools capable of generating tissue-like environments with experimentally defined and physiologically relevant oxygen gradients. Current 3D culture tools have not been widely adopted because they require specialized equipment and engineering expertise to set up, maintain, and analyze. These devices often do not interface well with current cell-based analyses such well plate end-point analyses or the selective removal and lysis of cells for molecular biology readouts (e.g., qPCR, Western blots, immunoassays). To enable the study of oxygen's role in directing responses at the cellular, tissue, and organ level, culture platforms that are readily accessible to laboratories who specialize in cell and molecular biology are needed. This MIRA proposal continues to innovate paper-based scaffolds as support for preparing tissue-like structures. This platform is unlike any other, employing readily available materials and simple technological solutions, to generate 3D cultures with defined extracellular environments, regardless of cell type or tissue structure. The level of experimental control afforded by the paper-based culture platform makes it a powerful enabling technology to probe cell-environment relationships in a spatially resolved manner. Using diffusion- dominated gradients, similar to those that form in healthy and poorly vascularized tissues, we are: 1) Developing tools to characterize the gradients that form in real-time, focusing on oxygen, pH, glucose, and lactate. 2) Evaluating differences between immotile and highly invasive cells, to determine the microenvironment's role?in particular, oxygen gradients?in promoting movement and drug resistance. 3) Generating tissue-like co-cultures with physiologically relevant oxygen gradients, with a specific focus on oxygen's role in liver zonation and hormone regulation in a breast lumen model. 4) Developing a platform to screen multiple 3D tissue structures in parallel. The ability to generate and evaluate many tissue structures in parallel will significantly improve screening processes to assess liver toxicity and identify potential endocrine disruptors.